James F. Catterall

7α-methyl-19-nortestosterone, a synthetic androgen with high potency: structure-activity comparisons with other androgens

CNNT. There was a good correlation between bioactivity and binding affinity to AR for the 7alpha-substituted androgens compared to T. In contrast, relative to their binding affinity to AR, the androgenic potency of DHT and 19-NT was lower compared to T. The reason for the lower in vivo androgenic activity of 19-NT is attributable to its enzymatic conversion to 5alpha-reduced-19-NT in the prostate. In the case of DHT, the lower bioactivity could be attributed to its faster metabolic clearance rate relative to T. The correlation was further investigated in vitro by co-transfection of rat ARcDNA expression plasmid and a reporter plasmid encoding the chloramphenicol acetyl transferase (CAT) gene driven by an androgen inducible promoter into CV-1 cells. All the androgens led to a dose-dependent increase in the CAT activity. MENT was found to be the most potent followed by DHT, 19-NT, T, and CNNT. The specificity of the androgenic response was confirmed by its inhibition with hydroxyflutamide, an antiandrogen. Thus, there was a good correlation between binding affinity and in vitro bioactivity in the transient transfection assay for the androgens. This suggests that the in vivo bioactivity of androgens could be influenced not only by binding affinity to receptors but also by factors such as absorption, binding to serum proteins and metabolism. However, the high potency of MENT is primarily related to its higher affinity to AR.

Expression of extracellular domain peptides of the FSH receptor and their effect on receptor-ligand interactions in vitro

Follicle-stimulating hormone receptor (FSH-R) displays considerable homology to luteinizing hormone receptor (LH-R) in structure and amino acid sequence. Comparison of the sequences of the extracellular domains (ECD) of the receptors reveals two regions (amino acids 4-56 and 265-319 in FSH-R) that share relatively little amino acid sequence similarity. This suggests that these variable regions may be important in providing specificity of ligand binding. We have expressed overlapping ECD peptides containing one or both of these regions (RFI, amino acids 5-125; RF2, amino acids 201-319; and RF3, amino acids 5-319) as fusion proteins in E. coli using pRSET vector. The presence of polyhistidine at the N-terminal end allowed substantial purification of the expressed proteins by a single step of affinity chromatography. The purified peptides were characterized for direct binding of hormone and their ability to block the binding of FSH and LH to the receptors. None of the peptides bound labelled hormone, while all peptides inhibited the binding of FSH to its receptor in a dose-dependent manner. However, only RF2 peptide inhibited ligand binding in a hormone-specific manner. These data suggest there is a site between amino acids 201-319 of the FSH-R ECD that is involved in FSH binding.

Cell-specific and hormonal regulation of the rat kidney androgen-regulated protein (KAP) gene

In mouse kidney, the kidney androgen-regulated protein (KAP) gene is regulated in a sex-dependent manner by a complex tissue- and cell-specific multihormonal system. KAP is also found in mouse uterus during the period surrounding birth. We describe here for the first time the existence of KAP in a species other than mouse. The rat cDNA sequence was determined and the derived peptide sequence displayed only 53% identity with murine KAP, although the genomic organization of the genes was identical. Expression of rat KAP was restricted to kidney and uterus, but was constitutive in the latter and drastically induced at parturition. The renal expression of the rat KAP gene was sexually dimorphic and regulated by physiological levels of steroid hormones. The effects of castration, hypophysectomy, thyroidectomy, and castration plus thyroidectomy on KAP mRNA levels in both kidney and uterus were determined. Constitutive expression of the protein was strictly dependent on thyroid hormone in female kidneys where it was modulated by estrogens and other ovarian factor(s). In the uterus, KAP mRNA was mainly under estrogen control. In males, expression of the KAP gene was under the dual regulation of thyroid hormone and androgens. Its complex regulation suggests a carefully delineated role for KAP in the kidney and uterus, but its physiological function remains unknown.

Identification and Characterization of an Androgen-responsive Kap Promoter Enhancer Located in the Intron II Region of human Angiotensinogen Gene

Transgenic expression of the human angiotensinogen (HAGT) gene directed by the mouse kidney androgen-regulated protein (Kap) gene promoter is proximal tubule cell-specific and androgen-regulated in vivo. The same Kap promoter fragment did not support similar regulation of other genes, but a transgene based on the original chimeric KAP-hAGT construct successfully directed NHE3 to kidney, suggesting that sequences within the HAGT gene fragment of the construct contributed to the regulation of its expression in vivo. In the present study, androgen-responsive regulatory sequences in the HAGT gene portions of the transgene were examined in transfected renal cells. A 1.4-kb enhancer between exons 2 and 3 was identified that increased the basal expression of Kap promoter 1.5- to 2-fold, its induction by dihydrotestosterone (DHT) 2- to 3-fold and its induction by dexamethasone (Dex) 4- to 5-fold. Sequence analysis revealed two potential hormone-responsive elements. Mutational assays and electrophoretic mobility shift assay showed one of these elements was androgen-specific. These findings may influence future strategies for the design of inducible, cell-specific transgenes.

Genetic variations in human testosterone-estradiol binding globulin

The human testosterone-estradiol-binding globulin (hTeBG) is a plasma heterogeneous glycoprotein with high affinity for a number of circulating steroid hormones. The heterogeneity originates from differential glycosylation of a common protein precursor. Analysis of desialylated hTeBG by isoelectric focusing (IEF) has revealed that microheterogeneity could be partly attributed to variability in sialic acid content or rearrangement of amino acid composition. We have studied this possibility by the analysis of desialylated serum hTeBG by Western blotting of proteins previously separated on IEF-gels. Two distinct well-defined IEF patterns were identified. The most frequent consisted of two major IEF-bands of equal color intensity. The other pattern consisting of four IEF-bands was present in only 5.55% of the total serum samples analyzed. Family studies showed that these phenotypes were autosomally inherited with a simple Mendelian transmission and allele frequencies had an excellent agreement between the observed and expected phenotypes. Androgen affinity constants and serum concentrations of hTeBG variant were similar to those of normal hTeBG. Molecular analyses of each of the exons of hTeBG gene by denaturing gradient gel electrophoresis revealed the presence of a point mutation in exon 8. The studies presented herein confirm and extend previous reports on the existence of structural variants of hTeBG. In addition, the mutation reported in this study is probably the same as that recently identified within numerous ethnic groups throughout the world, thus further supporting the concept of a two allele gene worldwide concoding hTeBG.

Identification of an attenuating region in the bovine follicle-stimulating hormone beta subunit mRNA that decreases its expression in E. coli.

Follicle-stimulating hormone (FSH) is one of the key regulators of gonadal function in mammals. Recombinant DNA expression of this hormone has proved to be a difficult task as expression levels are invariably low, irrespective of the expression system employed. In the present study, we have attempted to identify reasons for this low expression using bacterial expression vectors, and we report here the identification of a theoretically predicted hairpin structure in the mRNA corresponding to the N-terminal portion of the mature coding portion of bFSHbeta cDNA that is responsible for attenuating its expression in E. coli. When full-length FSHbeta was expressed using the bacterial expression vector, a very low expression was obtained. However, when fragments of FSHbeta with N-terminal deletions (amino acids 24-110 and 13-110) were expressed using the same expression strategy, a 30- to 40-fold higher expression was observed. This low expression of FSHbeta could be attributed to a hairpin structure present in the first 12 codons of mature FSHbeta mRNA. Disruption of this structure without changing the amino acid sequence resulted in a higher level of expression of FSHbeta. The predicted hairpin structure, though away from the transcriptional and translational start site, was able to downregulate the expression of FSHbeta probably by impeding the movement of ribosomes.